INFECTION AND IMMUNITY,0019-9567/99/$04.0010

Oct. 1999, p. 5434–5440 Vol. 67, No. 10

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Infection of Endothelial Cells with Trypanosoma cruzi Activates NF-kBand Induces Vascular Adhesion Molecule Expression

HUAN HUANG,1* TINA M. CALDERON,1 JOAN W. BERMAN,1,2 VICKI L. BRAUNSTEIN,1

LOUIS M. WEISS,1,3 MURRAY WITTNER,1 AND HERBERT B. TANOWITZ1,3

Departments of Pathology,1 Microbiology and Immunology,2 and Medicine,3

Albert Einstein College of Medicine, Bronx, New York 10461

Received 29 March 1999/Returned for modification 21 May 1999/Accepted 27 July 1999

Transcriptional activation of vascular adhesion molecule expression, a major component of an inflammatoryresponse, is regulated, in part, by the nuclear factor-kB/Rel (NF-kB) family of transcription factors. Wetherefore determined whether Trypanosoma cruzi infection of endothelial cells resulted in the activation ofNF-kB and the induction or increased expression of adhesion molecules. Human umbilical vein endothelialcells (HUVEC) were infected with trypomastigotes of the Tulahuen strain of T. cruzi. Electrophoretic mobilityshift assays with an NF-kB-specific oligonucleotide and nuclear extracts from T. cruzi-infected HUVEC (6 to48 h postinfection) detected two major shifted complexes. Pretreatment with 503 cold NF-kB consensussequence abolished both gel-shifted complexes while excess SP-1 consensus sequence had no effect. These dataindicate that nuclear extracts from T. cruzi-infected HUVEC specifically bound to the NF-kB consensus DNAsequence. Supershift analysis revealed that the gel-shifted complexes were comprised of p65 (RelA) and p50(NF-kB1). Northern blot analyses demonstrated both the induction of vascular cell adhesion molecule 1 andE-selectin and the upregulation of intercellular adhesion molecule 1 mRNA in HUVEC infected with T. cruzi.Immunocytochemical staining confirmed adhesion molecule expression in response to T. cruzi infection. Thesefindings are consistent with the hypothesis that the activation of the NF-kB pathway in endothelial cellsassociated with T. cruzi infection may be an important factor in the inflammatory response and subsequentvascular injury and endothelial dysfunction that lead to chronic cardiomyopathy.

Chagas’ disease, a consequence of infection with the hemo-flagellate parasite Trypanosoma cruzi, is a major cause of acuteand chronic myocarditis and cardiomyopathy in areas of ende-micity in Latin America (51). The pathogenesis of the cardiacdamage associated with this infection is multifactorial. Theclinical manifestations of T. cruzi infection may be the result offocal ischemia (13, 34, 50), autoimmune responses (18), anddirect parasite invasion of cells of the myocardium, all of whichmay promote myocardial inflammation (31, 54, 62). Recentstudies have underscored the primary role of inflammation inthe pathogenesis of chagasic heart disease. The inflammatoryresponse is composed of lymphocytes (predominately CD81)(46), monocytes, macrophages, and eosinophils. This inflam-matory process has been associated with the expression ofcytokines and inducible nitric oxide synthase (3, 16). In addi-tion, vascular adhesion molecules have been described both forthe heart and for sera obtained from infected mice and humans(19, 20, 31, 61). More recently, Sunnemark et al. (47) describedaortic vasculitis in T. cruzi-infected mice which was associatedwith inflammation and expression of cytokines and the adhe-sion molecule intercellular adhesion molecule 1 (ICAM-1).

The vascular endothelium is an important target of parasiteinvasion (48). T. cruzi infection of cultured endothelial cellsresults in expression and/or upregulation of important vasoac-tive molecules such as endothelin 1 (57, 59) and proinflamma-tory cytokines including interleukin-1b (IL-1b) and IL-6 (49,52, 59). Murine T. cruzi infection is also associated with circu-lating tumor necrosis factor alpha (TNF-a) (53) and throm-

boxane A2 (48). All of these factors have been implicated inT. cruzi-associated microvascular compromise.

Activation of the nuclear factor kB/Rel (NF-kB) family ofdimeric transcription factor complexes is regarded as an im-portant initial event in the vascular response to a variety ofinfectious agents (10, 14, 17, 36, 37, 43, 60), toxins, cytokines,growth factors, and oxidant stress (5, 6). The inactive formof the best-characterized NF-kB heterodimer, consisting of acomplex of p50 (NF-kB1) and p65 (RelA subunits), is retainedin the cytoplasm either by association with IkBa or by associ-ation of the p65 subunit with p105, a precursor of p50. Multiplesignal transduction pathways lead to phosphorylation, poly-ubiquitination, and degradation of IkBa or p105. Hetero-dimeric NF-kB enters and accumulates in the nucleus andcontributes to the transcriptional activation of many genesrelevant to endothelial pathophysiology, including those en-coding vascular adhesion molecules (27, 28, 29, 38, 56).

Modulation of the transcriptional activity of NF-kB is criti-cal to endothelial cell activation and the associated inflamma-tory response. Leukocyte accumulation at sites of local injuryor endothelial cell infection is dependent on the interaction ofcirculating leukocytes with vascular adhesion molecules. Theselectin family of adhesion molecules, including E-selectin (1,2), mediates the rolling and initial tethering of leukocytes tovascular endothelium while firm adhesion and transmigrationinto subendothelial tissue are mediated by members of theimmunoglobulin superfamily (44, 55), including vascular celladhesion molecule 1 (VCAM-1) (4, 12, 32, 33) and ICAM-1(45). NF-kB-like binding elements are present in the promoterregions of the E-selectin, VCAM-1, and ICAM-1 genes andplay a pivotal role in the transcriptional regulation of theseadhesion molecules (15, 25, 56). We therefore determinedwhether the inflammatory response elicited by T. cruzi infec-tion is characterized by NF-kB activation and induction or

* Corresponding author. Mailing address: Department of Pathology,Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx,NY 10461. Phone: (718) 430-2143. Fax: (718) 430-8543. E-mail:Huangh@aecom.yu.edu.

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increased expression of E-selectin, VCAM-1, and ICAM-1 inendothelial cells.

In the present study, we demonstrated that infection of cul-tured human umbilical vein endothelial cells (HUVEC) withT. cruzi is associated with activation of NF-kB and induction ofendothelial cell adhesion molecule expression. These studiesmay provide a cellular basis for the inflammatory response toT. cruzi infection that is important in the pathogenesis of cha-gasic cardiomyopathy.

MATERIALS AND METHODS

Infection and TNF-a treatment of endothelial cell cultures. Trypomastigotesof T. cruzi (Tulahuen strain) were harvested from the supernatants of infectedmyoblasts (35). HUVEC were isolated, cultured, and infected at a multiplicity ofinfection of 1.5 to 2.0:1 as previously described (49). The HUVEC had a char-acteristic cobblestone appearance and could be stained with antibody to vonWillebrand factor (DAKO Corporation, Carpinteria, Calif.). The percent para-sitism was determined by examination of fixed culture plates stained with May-Grunwald-Giemsa stain: parasitism was approximately ,1% at 1 to 6 h, 10% at24 h, 20 to 40% at 48 h, and .80% at 72 h postinfection. As a positive controlfor adhesion molecule expression and NF-kB activation, human recombinantTNF-a (Genzyme Diagnostics, Cambridge, Mass.) was added to uninfectedcultured cells at a final concentration of 100 U/ml.

Nuclear isolation and extraction. Extracts of infected and uninfected HUVECwere prepared as described by Read et al. (26). Briefly, cell monolayers (3 3 106

to 5 3 106 cells) were harvested by scraping, washed in cold phosphate-bufferedsaline (PBS), and incubated in 100 ml of buffer A (10 mM HEPES [pH 8.0], 1.5mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 200 mM sucrose, 0.5 mMphenylmethylsulfonyl fluoride, 1 mg of leupeptin per ml, 1 mg of aprotinin per ml,and 0.5% NP-40) for 10 min at 4°C. The crude nuclei released by lysis werecollected by microcentrifugation, and the nuclear pellet was rinsed once in bufferA and resuspended in 100 ml of buffer B (20 mM HEPES [pH 8.0], 20% glycerol,0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mMdithiothreitol, and 1 mg each of leupeptin and aprotinin per ml). Nuclei weresonicated for 10 s at 15% power output (Virsonic cell disrupter; Virtis, Gardner,N.Y.) and clarified by microcentrifugation for 30 s. The resulting supernatantscontained 1 to 2 mg of protein per ml by Bio-Rad assay (Bio-Rad, Richmond,Calif.) with bovine serum albumin as the standard. Nuclear extracts were frozenon dry ice and stored at 280°C.

Electrophoretic mobility shift (gel shift) assay. Assays were performed withthe gel shift assay system (Promega, Valencia, Calif.) according to the manufac-turer’s protocol, with 5 to 10 mg of nuclear protein. Sequences of double-stranded consensus oligonucleotides used in gel shift reactions were as follows:NF-kB (Promega), 59-AGT TGA GGG GAC TTT CCC AGG C-39; SP-1 (Pro-mega), 59-ATT CGA TCG GGG CGG GGC GAG C-39. Probe labeling wascarried out as specified by the manufacturer with [g-32P]ATP (3,000 Ci/mmol; 10mCi/ml) (Amersham, Arlington Heights, Ill.). Specificity studies were performedwith a 50-fold molar excess of unlabeled oligonucleotide added to the reactionmixtures prior to the addition of radiolabeled oligonucleotides. Reaction mix-tures were analyzed on 5% nondenatured polyacrylamide gels with 0.53 TBE(89 mM Tris-HCl [pH 8.0], 89 mM boric acid, 2 mM EDTA) as the runningbuffer. The gels were electrophoresed at 100 V for 3 h, dried (gel dryer), andsubjected to autoradiographic exposure for 12 to 48 h.

Electrophoretic mobility supershift assays. Polyclonal antibodies targeted top50 and p65 (100 mg/0.1 ml) were purchased from Santa Cruz Biotechnology,Inc. (Santa Cruz, Calif.). Prior to the addition of radiolabeled oligonucleotideprobe, 2 mg of antibody per gel shift reaction mixture was added and the mixtureswere incubated at room temperature for 20 min. Each reaction mixture wasanalyzed by gel shift assays as described above.

RNA preparation. HUVEC monolayers were washed briefly with cold PBSand immediately lysed with Trizol reagent. Total RNA was extracted as recom-mended by the manufacturer’s protocol (GIBCO BRL, Grand Island, N.Y.). Forprotection from RNase activity, the final RNA pellets were solubilized in Forma-zol (Molecular Research Center, Inc., Cincinnati, Ohio).

Northern blot analysis. A high-efficiency hybridization system was purchasedfrom Molecular Research Center, Inc. Northern blot analysis was performed asspecified by the manufacturer. Briefly, equal amounts of total RNA (15 mg) wereincubated in formaldehyde reaction solution at 55°C for 15 min and loaded onto1% agarose-formaldehyde gels. After electrophoresis, RNA was transferred tonitrocellulose filters. Filters were prehybridized at 42°C for 6 to 12 h and hy-bridized at 42°C for 24 to 48 h in high-efficiency hybridization solution withappropriate random-primed labeled denatured cDNA probes for humanVCAM-1, E-selectin (W. Newman, Otsuka Pharmaceuticals, Rockville, Md.),and ICAM-1 (Timothy Springer, Center for Blood Research, Harvard Univer-sity, Boston, Mass.) at 1 3 106 to 3 3 106 dpm/ml. Hybridization with 18S(rRNA) cDNA was utilized to verify the loading equivalency of each lane. Thefilters were washed, and autoradiography was performed with X-ray film and anintensifying screen at 270°C.

Immunocytochemistry. Fourth- or fifth-passage HUVEC were cultured ingelatin-coated 24-well plates for 3 days. Trypomastigotes were washed in PBS(pH 7.2) and resuspended in endothelial cell medium. Approximately 1.25 3 106

trypomastigotes were used to infect cells in each well. Supernatants from unin-fected myoblasts were used for sham infections. At 24 and 48 h postinfection, theplates were washed gently three times with PBS and then fixed for 8 min inice-cold methanol and 0.8% H2O2. After washing in PBS, wells were incubatedat room temperature with blocking solution (PBS–4% fetal bovine serum[GIBCO]) for 30 min. Primary antibodies in blocking solution were added to thewell and incubated overnight at 4°C. The primary monoclonal antibodies wereused at the following dilutions: mouse anti-human VCAM-1 (immunoglobulinG1 [IgG1]) (Becton Dickinson, San Jose, Calif.) (1:500), mouse anti-humanE-selectin or CD62E (ELAM-1) (IgG1) (1:500) (Becton Dickinson), humananti-ICAM-1 antibody (1:500) (DAKO Corporation), and anti-human von Wil-lebrand factor (IgG1) (1:100) (DAKO Corporation). In addition, purified IgG1mouse myeloma protein (Organon Teknika Corp., Durham, N.C.) was used as anisotype-matched negative control. Treatment of HUVEC with recombinant hu-man TNF-a (100 U/well) (Genzyme Diagnostics) was used as a positive controlfor the induction or increased expression of adhesion molecules. After incuba-tion with primary antibody, the wells were washed twice with PBS and incubatedwith biotinylated anti-mouse IgG (heavy plus light chains), avidin-biotin-coupledperoxidase (Vectastain ABC kit; Vector Laboratories, Inc., Burlingame, Calif.),and diaminobenzidine (peroxidase substrate kit; Vector Laboratories).

Statistical analysis. The results of Northern blot analyses of adhesion mole-cule expression by HUVEC were quantified by densitometry and normalized tothe corresponding 18S rRNA signal and expressed as a ratio. Data from threeseparate experiments were then analyzed by Student’s t test and were plotted asthe means 6 standard errors of the means.

RESULTS

T. cruzi infection of HUVEC upregulates or induces ICAM-1, VCAM-1, and E-selectin mRNA expression. Northern blotanalyses of adhesion molecule expression of HUVEC thatwere uninfected, TNF-a treated (24 h) (uninfected), and in-fected for 24 h are shown in Fig. 1. ICAM-1 was constitutivelyexpressed in untreated HUVEC and was upregulated in in-fected and TNF-a-treated cultures (Fig. 1, top panel). VCAM-1 mRNA expression was undetectable in uninfected cells andwas also significantly induced after infection with T. cruzi orTNF-a treatment (Fig. 1, middle panel). There was a signifi-cant increase in E-selectin mRNA expression in both T. cruzi-infected and TNF-a-treated HUVEC, while uninfected cellsexpressed a low basal level of E-selectin message (Fig. 1, bot-tom panel). These data indicate that T. cruzi infection ofHUVEC for 24 h upregulates or induces ICAM-1, VCAM-1,and E-selectin mRNA expression.

Time course of ICAM-1, VCAM-1, and E-selectin mRNAexpression by T. cruzi-infected HUVEC. Northern blot analysesof adhesion molecule expression by HUVEC that were unin-fected or infected for 6, 24, 48, and 72 h were performed. Datafrom three separate blots were quantified by densitometry,normalized to the 18S rRNA signal, and expressed as a ratio asshown in Fig. 2. Student t test analysis indicated that the threeadhesion molecules were significantly induced or upregulatedfrom 6 to 72 h postinfection (Fig. 2). ICAM-1, constitutivelyexpressed in uninfected cells, was upregulated from 6 to 72 hafter infection (Fig. 2A). VCAM-1 was induced after 6 h, andmessage levels increased up to 72 h postinfection (Fig. 2B), thelast time point analyzed. E-selectin expression was increased6 h postinfection and remained upregulated until 72 h (Fig.2C). These data suggest that infection of HUVEC by T. cruzicauses persistently elevated expression of adhesion molecules.

T. cruzi infection activates NF-kB in HUVEC. NF-kB wasassayed in nuclear extracts of infected HUVEC by electro-phoretic mobility shift assays with a 32P-labeled, double-stranded consensus NF-kB oligonucleotide corresponding tothe kB binding domain of the murine k light chain gene en-hancer (39). After 6 h of infection, two major gel-shifted pro-tein-DNA complexes were evident, a faster-migrating lowercomplex (S2) and an upper complex (S1). Both of these bands

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were negligible in uninfected HUVEC. A time course study re-vealed that these complexes were also evident in nuclear pro-tein extracts from HUVEC 24 and 48 h postinfection (Fig. 3).

To confirm the specificity of nuclear protein binding to theNF-kB oligonucleotide, competition assays were performed(Fig. 4). Nuclear protein extracts from HUVEC at 6 h postin-fection formed two major protein-DNA complexes (Fig. 4,lane 2) which were absent in uninfected cells (lane 1). Complexformation was specifically inhibited by incubation with a 50-fold molar excess of unlabeled NF-kB probe (Fig. 4, lane 3) butnot by treatment with an unrelated oligonucleotide containingthe SP-1 binding consensus sequence (lane 4). These dataindicate that T. cruzi infection of HUVEC induces nucleartranslocation of active NF-kB. To identify the subunit compo-sition of NF-kB in the protein-DNA complexes induced afterT. cruzi infection, supershift analyses were performed withpolyclonal antibodies specific for p65 and p50. Changes inelectrophoretic mobility resulting in supershifted complexeswere detected with the addition of anti-p65 and anti-p50 to thereaction mixtures prior to the addition of the labeled NF-kBoligonucleotide. Anti-p65 induced a supershift of the upperprotein-DNA complexes (Fig. 4, lane 5), while anti-p50 wasspecific for the lower protein-DNA complexes (lane 6). Non-immunized rabbit serum did not cause any supershifted com-plexes (Fig. 4, lane 7). These data suggested that the upper

complexes contain p65 and that the lower complexes containp50. TNF-a-treated (5 min) HUVEC nuclear protein extractwas used as a positive control and produced similarly shifted(Fig. 4, lane 8) and supershifted (lane 9, anti-p65, and lane 10,anti-p50) complexes.

T. cruzi infection induces or upregulates E-selectin, VCAM-1, and ICAM-1 protein expression. In order to examine theeffects of T. cruzi infection on cell surface expression of E-

FIG. 1. T. cruzi infection induces or upregulates ICAM-1, VCAM-1, andE-selectin mRNA. Northern blot analyses of adhesion molecule expression byHUVEC that were uninfected, TNF-a treated, or infected for 24 h are shown(see Materials and Methods). ICAM-1 mRNA was constitutively expressed inuntreated HUVEC and was upregulated in infected and TNF-a-treated cul-tures (top panel). VCAM-1 mRNA expression was undetectable in unin-fected HUVEC and was induced after infection or TNF-a treatment (middlepanel). There was a significant increase in E-selectin mRNA expression inboth T. cruzi-infected and TNF-a-treated HUVEC, while uninfected cells ex-pressed a low basal level of E-selectin message (bottom panel). An 18S rRNAprobe was utilized to normalize the total RNA loading equivalency of each lane.Data shown are representative of three separate experiments. Lane C, uninfect-ed cells as control; lane TNF-a, HUVEC treated with TNF-a; lane I, HUVECinfected with T. cruzi.

FIG. 2. Time course of ICAM-1, VCAM-1, and E-selectin mRNA expressionwith T. cruzi infection. Northern blot analyses of adhesion molecule expressionby HUVEC that were uninfected or infected for 6, 24, 48, and 72 h. Data fromthree separate blots were quantified by densitometry and normalized to the 18SrRNA signal and expressed as a ratio. Student t test analysis indicated that thethree adhesion molecules were significantly induced or upregulated from 6 to72 h postinfection (P , 0.001) (see Materials and Methods). (A) ICAM-1 wasupregulated from 6 to 72 h and was constitutively expressed in uninfected cells.(B) VCAM-1 was induced from 6 to 72 h postinfection. (C) E-selectin expressionwas increased at 6 h postinfection and remained upregulated until 72 h, the lasttime point analyzed.

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selectin, VCAM-1, and ICAM-1, monoclonal antibodies spe-cific for each adhesion molecule were used in an immuno-cytochemical analysis of infected HUVEC. Consistent with theresults of Northern blot analysis, E-selectin protein wassignificantly upregulated in both T. cruzi-infected (Fig. 5B)and TNF-a-treated (4 h) HUVEC (data not shown). Un-treated HUVEC were minimally reactive with the E-selectinantibody (Fig. 5A). Constitutive levels of ICAM-1 protein ex-pression (Fig. 5C) were also upregulated with T. cruzi infection(Fig. 5D). Similarly, VCAM-1 was induced by T. cruzi infection(Fig. 5F), whereas normal reactivity was detectable on unin-fected HUVEC (Fig. 5E). Untreated and infected HUVECexhibited no reactivity with the isotype-matched negative-con-trol IgG1 mouse myeloma protein (data not shown).

DISCUSSION

We demonstrated that T. cruzi infection of endothelial cellsis associated with activation of NF-kB and expression of theendothelial cell adhesion molecules E-selectin, VCAM-1, andICAM-1. These findings suggest a possible mechanism for therecruitment of circulating leukocytes, a critical factor in theinitiation of an inflammatory response, as a result of T. cruziinfection (50). The generation of an acute inflammatory re-sponse is important in the initial control of acute infection butmay be detrimental if it persists for prolonged periods and maycontribute to myocardial damage.

The vascular endothelium is an early target of T. cruzi inva-sion (48). We have demonstrated that infection of HUVECresults in alterations in host cell metabolism and signal trans-duction (23, 24, 48). In the murine model, infection causesvascular injury and altered function (13, 16, 49, 50, 52). Infec-tion and ensuing injury to the vascular endothelium, as well as

the ischemia-reperfusion damage to the heart, are importantfactors in the pathogenesis of chagasic heart disease. Amasti-gotes are not commonly found in endothelial cells in vivo onroutine histopathology. This is most likely due to issues oftiming and sampling.

In understanding the pathogenesis of Chagas’ disease, it isimportant to note that the infection is persistent throughoutthe lifetimes of patients and results in compromised microvas-culature including vasospasm and decreased blood flow. Dur-ing the chronic phase, antiparasitic therapy usually fails toattenuate the progression of the disease. Therefore, on thebasis of observations made from experimental infections andhuman disease, we and others hypothesized that after the ini-tial acute insult to the endothelium, endothelial cells undergodamage and regeneration. In other settings such as balloonangioplasty-induced endothelial removal (41) and Kawasakidisease (7), regenerated endothelial cells no longer functionnormally, resulting in endothelial dysfunction and cardiac pa-thology. Similarly, we believe that the upregulation of the in-flammatory process and the subsequent ischemia after T. cruziinfection contribute to the development of chronic chagasiccardiomyopathy.

The mechanism of T. cruzi-associated expression of vascularadhesion molecules remains to be defined. We demonstratedpreviously that T. cruzi infection of HUVEC results in theexpression of IL-1b and IL-6 (49). These cytokines are knownto induce expression of vascular adhesion molecules. However,the primary event necessary for expression of vascular adhe-sion molecules requires the activation of NF-kB, which alsoplays a critical role in cytokine gene transcription and transla-tion. Lockyer et al. (21) demonstrated that inhibition of NF-kBafter transfection of a mutated IkB gene into human endothe-

FIG. 3. T. cruzi infection activates NF-kB in HUVEC. NF-kB was assayed innuclear extracts of HUVEC by electrophoretic mobility shift assays with a 32P-labeled, double-stranded consensus NF-kB oligonucleotide. Lanes 1, 3, and 5represent nuclear extracts obtained from uninfected HUVEC after 6, 24, and48 h in culture, respectively. Lanes 2, 4, and 6 represent nuclear extracts obtainedfrom HUVEC infected for 6, 24, and 48 h, respectively. Two shifted complexesappeared in the nuclear protein-DNA interaction from infected HUVEC (S1,shifted complex 1; S2, shifted complex 2). Both shifted complexes were unde-tectable in uninfected HUVEC. The arrow at the bottom indicates free NF-kBprobe.

FIG. 4. Identification of the shifted complexes as a result of T. cruzi infection.To confirm the specificity of nuclear protein binding to the NF-kB oligonucle-otide, competition assays were performed. Nuclear extracts from HUVEC at 6 hpostinfection formed two major protein-DNA complexes (S1 and S2) (lane 2)which were absent in uninfected HUVEC (lane 1). Complex formation wasspecifically inhibited by incubation with a 50-fold molar excess of unlabeledNF-kB probe (lane 3) but not by a 50-fold molar excess treatment with anunrelated oligonucleotide containing the SP-1 binding consensus sequence (lane4), indicating that these nuclear proteins specifically bound to the NF-kB con-sensus sequence. To identify the subunit composition of NF-kB in the protein-DNA complexes induced after infection, supershift (SS) analyses were per-formed with polyclonal antibodies specific for p65 and p50. Anti-p65 caused asupershift of S1 (lane 5), while anti-p50 induced a supershift of S2 (lane 6).Nonimmunized rabbit serum did not cause any supershifted complexes (lane 7).TNF-a-treated HUVEC nuclear protein extract was used as a positive controland produced similarly shifted (lane 8) and supershifted (lane 9, anti-p65 super-shift; lane 10, anti-p50 supershift) complexes. Data are representative of threeseparate experiments. The arrow at the bottom indicates free NF-kB probe.

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lial cells blocked the expression of adhesion molecules in re-sponse to TNF-a. Morishita et al. (22) introduced syntheticdouble-stranded oligodeoxynucleotides into rat hearts to blockthe nuclear translocation of NF-kB and found that the expres-sion of cytokines and adhesion molecules was effectively inhib-ited during the ischemia-reperfusion event, thereby reducingthe extent of myocardial infarction. Transfection of the sameoligodeoxynucleotide into human endothelial cells also inhib-ited the expression of cytokines and adhesion molecules. Thesedata indicate that NF-kB plays a pivotal role in the initiation ofan inflammatory response. We believe that T. cruzi infection ofHUVEC induces the activation of NF-kB, which leads to the

production of IL-1b and IL-6. These cytokines also cause apositive feedback effect, thus further activating NF-kB.

Many extracellular signals trigger the activation of NF-kBby a number of signal transduction pathways. For example,TNF-a binds to its receptors and initiates second messengerand signaling cascades (42), resulting in the activation of theIkB kinase complexes including IKKa and IKKb (8, 30, 61).These kinases are necessary for IkB phosphorylation and deg-radation and subsequent NF-kB activation. How parasite-en-dothelial cell interactions activate signaling pathways involvedin NF-kB activation is unknown. The interaction and infectionof endothelial cells with Rickettsia rickettsii also activate NF-kB

FIG. 5. T. cruzi infection induces or upregulates E-selectin, VCAM-1, and ICAM-1 protein expression. Shown are photomicrographs of adhesion protein expressionin infected HUVEC as demonstrated by immunocytochemistry. Uninfected HUVEC incubated with isotype-matched, negative-control, purified IgG1 mouse myelomaprotein did not exhibit nonspecific staining (see Materials and Methods). Bar, 150 mm. (A) Uninfected HUVEC stained with anti-E-selectin antibody. There is minimalbackground staining and E-selectin expression. (B) HUVEC infected for 24 h and stained with anti-E-selectin antibody. E-selectin protein was upregulated. (C)Uninfected HUVEC stained with anti-ICAM-1 antibody. Cells constitutively expressed ICAM-1 protein. (D) HUVEC infected for 24 h and stained with anti-ICAM-1antibody. ICAM-1 protein expression was upregulated. (E) Uninfected HUVEC stained with anti-VCAM-1 antibody. There is minimal VCAM-1 expression. (F)HUVEC infected for 24 h and stained with anti-VCAM antibody. VCAM-1 protein was upregulated.

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(43) and induce the expression of adhesion molecules (9, 40).This organism shares many of the characteristics of T. cruzi,since they both invade and reside in endothelial cell cytoplasm.Currently, we are exploring alterations in intracellular signal-ing cascades during T. cruzi infection. The data presented inthis report demonstrate that NF-kB is continuously activatedfrom 6 to 48 h postinfection, indicating that parasitism canactivate this pathway for extended periods. In addition, we alsofound that T. cruzi infection of endothelial cells (24) and vas-cular smooth muscle cells (23) activates phospholipase C, partof the signaling pathways involved in NF-kB activation (58).Infection with this parasite may also activate other kinases inhost cells and cause the subsequent phosphorylation of the IkBsubunit. Furthermore, T. cruzi is rich in secretory proteases(11) which may degrade the IkB subunit, thereby directly ac-tivating the NF-kB pathway. Currently, these cellular eventsare under investigation in our laboratory.

ACKNOWLEDGMENTS

This work was supported by a New Investigator Development Awardfrom the American Heart Association, New York City Affiliate (H.H.);grants-in-aid from the American Heart Association (J.W.B. and H.H.);and Public Health Service grants AI-12770 (H.B.T.) AI-39454(L.M.W.), and AI-41752 (M.W.).

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